Sigma-Aldrich Complete His-Tag Purific.

Form

suspension

Packaging

pkg of 200 mL (05893801001 [settled resin volume])
pkg of 25 mL (05893682001 [settled resin volume])

Manufacturer/Tradename

Roche

Technique(S)

protein purification: suitable

Matrix

Sepharose-CL 6B

Storage Temp.

2-8°C

Related Categories

Roche® Life Science Products

General Description

Bead size:45 to 165μM

Binding capacity:≥ 40mg protein per mL bed volume of resin. The binding capacity of the resin to various types of proteins may vary according to the protein characteristics such as the size of the protein. cOmplete His-Tag Purification Columns bind with a high specificity to the polyhistidine-tagged protein. As a consequence, the binding kinetics may appear to be different when compared to conventional metal chelate matrices. Full capacity of cOmplete His-Tag Purification Columns can be achieved by allowing more time for the protein to bind to the resin by lowering the flow rate during the chromatography purification procedure.

Maximal linear flow rate:1,420cm/hour

Recommended volumetric flow rate:The volumetric flow rate is a function of the cross section of the column. Using the following formula, a linear flow rate can be converted to a volumetric flow (mL/min.): Linear flow rate (cm/hour) × column cross sectional area (cm²)/60. The column cross sectional area is defined as Π× r², whereas Π is the constant pi and r is the inner radius of the column.

Recommended imidazole concentration for load/wash:Nonspecific binding of proteins without a His-tag is low. Use up to 5mM imidazole in load and/or wash buffers. If establishing a new assay for purification of His-tag proteins with cOmplete His-Tag Purification Columns, do not use imidazole. If, e.g., the purity of the His-tag protein needs to be improved following this first step, use imidazole in a final concentration of up to 5mM in a second step.

Recommended imidazole concentration for elution:Up to 500 mm. Please note: Contrary to other available resins, bound His-tagged protein typically elutes from cOmplete His-Tag Purification Columns with a lower imidazole concentration, e.g., 25 to 45mM.

Compatibility for long term storage:20% ethanol, pH 4.0 to pH 9.0

Compatibility during chromatography:The resin is compatible with 10mM EDTA, 10mM DTT during the purification (1 hour incubation), 6M guanidinium-HCl, 8M urea, pH 2.0 to pH 14.0.

Compatibility during cleaning:4% SDSThe cOmplete His-Tag Purification Resin is an innovative high-capacity IMAC matrix (Immobilized Metal Affinity Chromatography) for the purification of histidine-tagged proteins via batch or liquid chromatography procedures from total lysates. In contrast to all currently available resins, which are primarily based on NTA or IDA chelator chemistry, this resin is the only resin on the market which tolerates buffers that contain EDTA. Because EDTA is a known inhibitor of metalloproteases–which are frequently present in any cell type–this chromatography material provides the option to increase the protection of your target protein by supplementing your buffers with EDTA. This resin therefore allows increased protection of your proteins against degradation and, at the same time, enriches the protein using the well-established affinity purification method.
In addition, the resin maintains its binding capacity whether you are using low- or high-molecular weight proteins, and its specificity allows a one-step purification. By using one of the strongest chelators available, minimal contamination of your flow-through fractions with metal ions is ensured, safeguarding your downstream applications.
The cOmplete His-Tag Purification Resin is also available as prepacked format: with 1mL or 5mL resin.

Application

Recombinant Protein Expression

Purifying a protein of interest is often essential for determining its function, structure, or interactions, for raising specific antibodies, or preparing enzymes for practical applications. Isolation of naturally expressed proteins from their original source can be a complex process involving numerous chromatographic steps. Recombinant protein expression in dedicated host organisms can greatly simplify this task. Such expression systems generally ensure higher expression levels. Fusing the target protein to a tag also confers advantageous binding ability to an affinity matrix.

Protein Purification using Immobilized Ni²⁺

The most common technique in affinity purification of proteins involves engineering a sequence of 6 to 14 histidines into the N- or C-terminal (or even on an exposed loop) of the protein. Such polyhistidine stretches bind strongly to divalent metal ions such as nickel and cobalt. This effect can be used to separate proteins. Metal ions can be immobilized on a matrix using a chelator, which still allows the ion to interfere with the polyhistidine tag of the protein. When these his-tagged proteins are passed through a column containing immobilized metal ions, the proteins bind via the tag to the column. Nearly all untagged proteins pass through the column. The protein is released from the column by elution with either imidazole, which competes with the polyhistidine tag for binding to the column, or by a decrease in pH, which decreases the affinity of the tag for the resin. While this procedure is generally used for the purification of recombinant proteins with an engineered affinity tag, it can also be used for natural proteins with an inherent affinity for divalent cations.

His-Tags

Ideally, the His-tagged target protein binds much stronger to the Ni²⁺chelate matrix than endogenous histidine-containing protein of the expression host. Relative binding strength depends on how many histidines can bind simultaneously to the matrix (avidity effect). Longer His-tags confer stronger binding and better separation of the target from potentially contaminating host proteins. The classic His-tag has six consecutive histidines. Tags with 10 to 14 histidines may produce a better purification. Most importantly, His-tagged proteins can be purified using Ni²⁺chelate matrices under both native and denaturing conditions. Due to their hydrophilic and flexible nature, these matrices increase the solubility of the target proteins and only rarely interfere with protein function. This unique combination of features enables the His-tag to be a versatile tool for a wide range of protein purification applications.

Features And Benefits

cOmplete His-Tag Purification Resinis the only resin for purifying large amounts of protein without compromises.

• Use the buffer conditions best suited to your protein:Keep your protein comfortable and let it, not your purification resin, determine whether you use DTT, EDTA, or other buffer substances.
• Repeatedly obtain highly pure protein:Single step purification without resin recharging.
• Protect your protein from toxic nickel:Reduce protein oxidation and aggregation caused by resins that leach nickel.
• Work in a safe and eco-friendly environment:Avoid handling of toxic nickel and completely eliminate disposal costs.

Physical Form

50% resin in suspension, pre-charged with Ni²⁺

Other Notes

EDTA-compatible resin for the purification of poly-histidine-tagged proteins.For life science research only. Not for use in diagnostic procedures.cOmplete is a trademark of RocheGHS02,GHS07,GHS02H226 - H319 - H226P210 - P280 - P210 - P233 - P240 - P241 - P242 - P243 - P303 + P361 + P353 - P337 + P313 - P370 + P378 - P403 + P235